orientation of the vesicles does not have an impact on the experiment itself, but only on its interpretation. However, while theoretically possible through loading of vesicles, this experimental
protocol is not suitable to investigate export from vesicles.
2 Materials
2.1 Yeast Strains
for the Expression
of ABC and MATE
Tranporters
Saccharomyces cerevisiae strains (ura3-defective): YPH 499 (MATa
ura3-52 lys2-801 ade2-101 trp1-D63 his3-D200 leu2-1); W303
(MATa/MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his311,15} [phi+]); YBT1 (MATa; ura3Δ::HIS3; leu2-3, 112; his3-Δ
200; bat1Δ1::URA3) (see Note 2).
2.2 Growth Media
for S. cerevisiae Cells
– YPD medium: 1% Bacto yeast extract, 2% Bacto peptone, 2%
glucose. Autoclave 121
C, 20 min.
– Selective SD medium: 0.1% drop mix (amino acids and adenine
mix, see below), 0.67% YNB (Yeast Nitrogen Base without
amino acids and ammonium sulfate), 2% glucose. Autoclave
121
C, 20 min. Once autoclaved add (this is for example the
case of expressing vectors that harbor the gene ura3): 100Â His
(5 mL in 500 mL media), 100Â Leu (5 mL in 500 mL media),
100ÂTrp (5 mL in 500 mL media) (see below).
– Drop mix:
Final concentration (mg/L)
g per 14.5 g mix
Adenine
40
0.4
L-arginine
20
0.2
L-tyrosine
30
0.3
L-isoleucine
30
0.3
L-phenylalanine
50
0.5
L-glutamic acid
100
0.1
L-aspartic acid
100
1.0
L-threonine
200
0.2
L-serine
400
4.0
L-valine
150
1.5
L-methionine
150
1.5
L-lysine
180
1.8
– 100Â Ura: in 100 mL bidistilled water (bd H 2 O) dissolve 0.4 g
of L-uracil (0.8 g/200 mL).
92
Olivia Costantina Demurtas et al.